|
| Status |
Public on Sep 22, 2010 |
| Title |
TS13/4F-32-6 |
| Sample type |
RNA |
| |
|
| Source name |
human iPS cells from neonatal fibroblast BJ using SeV/TS13-MYC, -OCT4, -SOX2 and KLF4.
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: iPS cells
gender: male
age: 0y
|
| Treatment protocol |
Human fibroblasts (BJ, HDF) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum in a humidified incubator with 5% CO2. Human fibroblasts were then infected with SeV vectors carrying OCT4, SOX2, KLF4 and c-MYC at an MOI of 3 for overnight in DMEM supplemented with 10% heat-inactivated fetal bovine serum in a humidified incubator with 5% CO2. The medium was exchanged on the next day and cultured for 6 days in the same medium described above. Then the cells were collected by trypsin treatment, counted and then transfered at 1 x 10^5 cells per 10 cm dish onto MMC-treated MEF feeder cells. The next day, medium was exchanged to primate ES medium supplemented with 4 ng/ml bFGF. Cells were cultured and when ES-like colonies were appered (iPS cells), the colonies were picked up and cultured on MEF feeder cells. After more than 10 passages, the iPS cells were confirmed both that they expressed human ES marker genes by RT-PCR and SeV genome was not detected by quantitative RT-PCR.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using the RNeasy Mini Kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 50ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 50ul of Agilent 2x HI-RPM Hybridization Buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565B) using one color scan setting for 4x44k array slides (Scan Area 75x25 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 10-100% (XDR) ).
|
| Description |
Gene expression of TS13/4F-32-6 iPS clone at passage 10
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software v 9.5.3.1 (Agilent) using default parameters (protocol GE1-v5_95 and Grid: 014850_D_F_20100115) to obtain background subtracted and spatially detrended Processed Signal intensities.
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| |
|
| Submission date |
Sep 21, 2010 |
| Last update date |
Sep 21, 2010 |
| Contact name |
Noemi Fusaki |
| Organization name |
DNAVEC Corporation
|
| Department |
Cell Therapy & Regenerative Medicine
|
| Street address |
6 Ohkubo
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
300-2611 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE24240 |
Viral and transgene-free human induced pluripotent stem (iPS) cells using non-integrating Sendai virus (SeV) vectors. |
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